For preventing disease caused by annual epidemics of viral infections, vaccination is the most important public health measure. An effective supply of vaccines is dependent on being able to quickly produce large quantities of vaccine material (e.g. virus). The rapid development of vaccines and their abundant availability is critical in combating many human and animal diseases. Delays in producing vaccines and shortfalls in their quantity can cause problems in addressing outbreaks of disease.
Growth of viruses, especially of influenza virus in embryonated chicken eggs, has been shown to result in effective production of influenza virus particles which can be either used for production of inactivated or live attenuated influenza virus vaccine strains. Nevertheless during the last few years intensive efforts have been made in establishing virus production systems using cell culture because an egg-based method requires a steady supply of specific pathogen-free eggs which could be problematic in case of a pandemic. The cell-based technology is an alternative production process that is independent of eggs suppliers and can be started as soon as the seed virus is available. Besides this, inactivated influenza vaccine prepared from the virus grown in mammalian cells was shown to induce more cross-reactive serum antibodies and reveals better protection than egg-grown vaccine (Alymova et al., 1998, J Virol 72, 4472-7). Moreover, according to previous results receptor specificity and antigenic properties of human isolates become altered following growth of the virus in embryonated chicken eggs (Mochalova et al., 2003, Virology 313, 473-80, Romanova et al., 2003, Virology 307, 90-7).
On the other hand, multiple propagation of viruses in tissue culture often results in HA mutants that have elevated pH of fusion (Lin et al., 1997, Virology 233, 402-10) which is correlated to decreased stability to thermal denaturation of viruses (Ruigrok et al., 1986). The structure of any protein and its stability are based on noncovalent interactions like hydrophobic forces, van der Waal interactions, hydrogen bonds, and ionic interactions. Mutations which appear upon adaptation of viruses to cell cultures are known to elevate the threshold of pH of fusion induced by reduced protein stability because of changed ionic interactions and salt bridges in HA molecule (Rachakonda et al., 2007, Faseb J21, 995-1002). Destabilizing mutations usually found either at the interface HA1-HA2 or HA2-HA2 regions or in the N terminus of HA2 in turn could lead to reduced binding to cell-surface receptors (Korte et al., 2007, Rachakonda et al., 2007, Faseb J 21, 995-1002, Shental-Bechor et al., 2002, Biochim Biophys Acta 1565, 81-9), which leads to decreased virus infectivity and subsequently reduced immunogenicity of live virus preparations.
Massaab (Massaab H. F., Journal of Immunology. 1969, 102, pp. 728-732) tested the biologic and immunologic characteristics of cold-adapted influenza virus using different genetic markers before and after adaptation to growth in primary chick kidney tissue culture and embryonated eggs. It is stated that these strains are more sensitive to low pH compared to original “wild type” strains and showed marked decrease in infectivity and hemagglutination yields.
Fiszman et al (Journal of Virology, 1974, 13, pp. 801-808) examined the effect of low pH (pH6.6) on vesicular stomatitis virus (VSV) and showed that no viral particles or nucleocapsids were detected. Ackermann W and Massaab H. F. (Journal of Experimental Medicine FEB 1954, 99, pp 105-117) disclosed the effect of a viral inhibitor, alpha-amino-p-methoxy-phenylmethanesulfonic acid) upon the growth cycle of influenza virus.
Due to difficulties in obtaining high amounts of vaccine virus preparations from cell culture which are of high stability and immunogenicity in order to avoid any safety or supply issues, it is an object of the present invention to make available processes which lead to efficacious and stable viruses. The object is achieved by the provision of the embodiments of the present application.
The invention relates to a method for producing pH stable enveloped viruses in tissue cultures by employing conditions of decreased pH during dilution of virus suspension and host cell infection. The method of the invention also provides virus of increased stability and immunogenicity compared to virus particles derived from presently used methods.